Aplastic anemia (AA) and other types of bone marrow failure have clinical and laboratory features consistent with an autoimmune pathophysiology, with a diversity of putative inciting antigens, including viruses, chemicals, medical drugs, and tumor antigens. Whatever its specific etiology, a majority of patients respond with hematologic improvement after immunosuppressive therapies. One important clinical feature of AA is its association with stem cell clonal hematologic diseases, especially paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndromes (MDS). Accrual has been terminated for a research study of 100 patients with severe AA on presentation that added mycophenolate mofetil, as well as delayed addition of cyclosporine, to standard ATG, in an effort to induce tolerance. Hematologic response rates at 3 and 6 months were comparable to those achieved by standard treatment; relapse, evolution, and survival analyses are underway. Current protocols randomize previously untreated severe AA patients to either ATG plus cyclosporine for two years (long course immunosuppression) or to ATG, cyclosporine and rapamycin for six months (more intensive early treatment). For refractory disease, patients are randomized to treatment with either rabbit ATG or CAMPATH-1, a monoclonal antibody to T cells. Daclizumab, a monoclonal antibody that binds to the interleukin-2 receptor and has relative specificity for activated T cells, has been successful in about 30% of patients with moderate AA, and is now being applied to early relapse of severe AA (without profound neutropenia) and in single lineage marrow failure sates, pure red cell aplasia and amegakaryocytic thrombocytopenic purpura. In the laboratory, efforts have concentrated on the incitement of AA by an unknown virus (see Z01 HL 02319-14 HB), the aberrant immune response, and the problem of clonal evolution. In efforts to more specifically characterize the immune response, we have utilized the methods of flow cytometry and spectratyping to determine expansion of V-beta TCR families of T cells and skewing of CDR3 expression within expanded families, as indicators of antigen-driven clonal T cell proliferation. We are exploiting CD8 cytotoxic lymphocyte oligoclonal expansions detection of individual CDR3 sequences, evidence of an antigen-driven immune response, to monitor patients during and after theapy, especially as predictors of hematologic relapse. We have employed DNA chip analysis to examine CD34 hematopoietic cells, both normal and from patients with marrow failure syndromes, first for AA and subsequently for cytogetically defined myelodysplastic syndromes that commonly evolve from immune marrow failure (trisomy 8 and monosomy 7) and the clonal disease paroxysmal nocturnal hemoglobinuria (PNH), closely associated with AA. In PNH, paired primary CD34 cells from individual patients show marked upregulation of immune and apoptosis genes in the abnormal putative PIG-A-negative population; the PNH clone pattern was close to normal, and additionally there was little difference in the transciptome of CD34 cells obtained from patients with predominantly hemolytic compared to marrow failure clinical variants. In MDS, specifically up- and down-regulated genes have been observed for CD34 cells from patients with well-defined cytogenetic abnormalities in myelodysplasia . Trisomy 8, but not monosomy 7, also is associated with immune abnormalities related to hematopoiesis, as cytogenetically abnormal cells are more likely than normal cells to express Fas and to be apoptotic. Aneuploid cells in trisomy 8 are apototic, as they express Fas and annexin. The pattern of T cell usage in trisomy 8 resembles AA. Isolated CD8 T cell clones show preferential activity against cytogenetically abnormal cells, suggesting that they are reactive to a partially transformed clone of hematopoietic cells. Monosomy 7, a syndrome usually associated with aa fatal course due to refractory pancytopenia or acute leukemia, chromosomally aberrrant cells are abnormally sensitive to G-CSF in vitro, and high concentrations of this cytokine appear to select for pre-existing minor populations of monosomy 7 cells only detectable by the sensitive fluorescent in situ hybridization methodology. Molecular mechanisms for both trisomy 8 and monosomy 7 have been explored in the laboratory. For trisomy 8, oligoclonal T cell responses appear to be directed to the aneuploid clone, but cells are salvaged from fully undergoing cell death as result of blocked apoptosis; a putative antigen, suggested by the micoarray studies, the gene product of WT1, is overexpressed in this form of MDS and may target the abnormal clone. In monosomy 7, which has been associated with prolonged neutropenia and treatment with granulocyte colony stimulating factor (G-CSF), a truncated version of the G-CSF receptor, which signals proliferation by not differentiation, is present in the aneuploid cells and may be selected under conditions of high endogenous G-CSF or adminstered cytokine. In a mouse model of AA based on infusion of parental lymph node cells into F1 recipients, a prominent "innocent bystander" mechanism of hematopoietic stem cell killing has been demonstrated in co-transplantation experiments, likely explaining some of the extraordinary potency of limited numbers of activated T cells in human marrow failure diseases. We have implicated genes of the telomerase repair complex, which acts to preserve telomere length at mitosis in mammalian cells, in late onset AA and MDS. Two genese, TERC (for the RNA component) and DKC1, cause the constitutional AA disease called dyskeratosis congenita. In studies of kindreds with probands presenting with apparently acquired AA, without physical stigmata but with other family members showing mild hematologic abnormalities, we identified two novel mutations in TERC in all affected members. Despite only mild anemia, erythrocyte macrocytosis, or thromboycotopenia, marrows were strikingly hypocellular and showed low content of CD34 cells and functional colony-forming progenitor cells. One family member had been misdiagnosed with MDS at an advanced age, and both he and the proband showed good therapeutic response to instition of androgens (frequently effective in inherited marrow failure syndromes). Multiple different mutations in the TERT gene for the telomerase enzyme itself, not previously associated with disease in humans, in patients with onset of marrow failure in middle-age. In an AA additional patient with a deletion in TERC, and a strikingly positive family history of marrow disease, telomere length was normal but telomere single strand overhang was markedly eroded; telomere overhang shortening may precede telomere shortening. Histocompatability antigens and cytokine promoter polymorphisms have been suggested as immune system risk factors for AA; we now propose TERC and TERT abnormalities as responsible, through acclerated telomere shortening, for greatly diminished stem cell compartments and therefore hematopoietic system risk factors for marrow failure. In vitro experiments also suggest that telomerase activity may be modulated by androgens, providing a mechanism for the utility of male hormones in constitutional marrow failure states. Finally, studies of mitochondrial DNA sequence of CD34 cells in normal individuals and in MDS have been extended to single cell assays for CD34 cells, T cells, B cells and granulocytes; this methodology may prove useful in tracking stem cells, measuring the mammalian mutation rate, and to detect minimal residual malignant disease.